Methods and compositions for isolating small rna

ABSTRACT

The present invention concerns the use of methods and compositions for the isolation of small RNA molecules (100 nucleotides or fewer), such as microRNA and siRNA molecules. Such molecules are routinely lost in commonly used isolation procedures and therefore the present invention allows for a much higher level of enrichment or isolation of these small RNA molecules.

BACKGROUND OF THE INVENTION

The present application is a continuation of U.S. application Ser. No.14/886,682 filed Oct. 19, 2015, which is a division of U.S. applicationSer. No. 13/778,952 filed Feb. 27, 2013 (now U.S. Pat. No. 9,193,748),which is continuation of U.S. application Ser. No. 12/610,807 filed Nov.2, 2009 (now U.S. Pat. No. 8,404,439), which is continuation of U.S.application Ser. No. 10/667,126 filed Sep. 19, 2003 (abandoned), whichapplication claims the benefit of U.S. Provisional Application No.60/490,325 filed Jul. 25, 2003, which applications are incorporated byreference herein in their entirety.

1. Field of the Invention

The present invention relates generally to the fields of molecularbiology and biotechnology. More particularly, it concerns methods andcompositions for isolating small RNA molecules that are typically 100nucleotides or fewer, such as siRNA and miRNA, as opposed to larger RNAor DNA molecules. The isolated small RNA molecules can be used insubsequent studies or assays.

2. Description of Related Art

The study of small RNAs-RNA molecules on the order of 100 nucleotides orfewer—from various tissues in many organisms, as well as cultured cells,is an area of extreme interest now, and promises to remain one for thefuture. These small RNAs include microRNA molecules (miRNA) and smallinterfering RNA molecules (siRNA), both of which can have a powerfuleffect on the expression of a gene by virtue of hybridization to theirtarget mRNA. Additionally, these procedures would be applicable toisolating small nuclear and small nucleolar RNAs (snRNAs and snoRNAs),involved in mRNA and rRNA processing. The procedures could also be usedto isolate tRNAs along with 5S and 5.8S rRNAs, which are all involved inprotein translation.

Key to these studies is the need to isolate RNA molecules in the sizerange of 15 to 100 nucleotides with high efficiency. Methods thatprovide a straightforward methodology to do this are therefore quitevaluable.

The preparation of RNA from natural sources (tissue samples, wholeorganisms, cell cultures, bodily fluids) requires removal of all otherbiomolecules. Once water is eliminated, the primary component of cellsis usually protein, often providing three-quarters of the mass. Of themajor other biomolecules, lipids, carbohydrates, combinations of thesewith each other and protein, and DNA are the other main components. Agoal of RNA extraction is to remove protein and DNA, as these providethe greatest interference in the use of RNA. Lipid and carbohydratemoieties can usually be dissolved away with the aid of a detergent.Protein can be stripped off RNA (and DNA) with the aid of detergents anddenaturants, but still must be removed from the common solution.

Two main methods have historically been used to accomplish this end. Thefirst is the use of organic solvents that are immiscible with water todissolve (literally, to chemically extract) or precipitate proteins,after which the aqueous, protein-free phase can be separated bycentrifugation prior to removal. Usually, phenol or phenol-chloroformmixtures are used for this purpose. The second method selectivelyimmobilizes the RNA on a solid surface and rinses the protein away,after which conditions are used to release the RNA in an aqueoussolution. This is literally a solid-phase extraction. Both procedurescan reduce the amount of DNA contamination or carryover, with theefficiency varying with the precise conditions employed.

Phenol and phenol-chloroform extractions provide an extremely protein-and lipid-free solution of nucleic acid. Much if not all (depending onthe sample) of the carbohydrate is also lost in this procedure as well.Acid phenol-chloroform is known to extract some of the DNA out of theaqueous solution (Chomczynski and Sacchi, 1987). However, the solutionis high in denaturing agents such as guanidinium hydrochloride,guanidinium thiocyanate, or urea, all of which are incompatible withdownstream enzymatic analysis, and the first two with electrophoreticanalysis as well. RNA is usually separated from these mixtures byselective precipitation, usually with ethanol or isopropanol. Thisprocedure is not as effective for small nucleic acid molecules, so thisprocedure is not ideal for the preparation of small RNAs.

Solid-phase extraction relies on high salt or salt and alcohol todecrease the affinity of RNA for water and increase it for the solidsupport used. The use of glass (silica) as a solid support has beenshown to work for large RNAs in the presence of high concentrations ofdenaturing salts (U.S. Pat. Nos. 5,155,018; 5,990,302; 6,043,354;6,110,363; 5,234,809; Boom et al., 1990) or lower concentrations ofdenaturing salts plus ethanol (U.S. Pat. No. 6,180,778). However, normalconditions for binding to glass fiber for RNA do not work for microRNA,and the use of a raw lysate is problematic due to variable requirementswith different tissues.

Many of the protocols known involve isolation of DNA or larger mRNA,which are not ideal for isolation of small RNA molecules because theseare often not effectively captured and eluted. Thus, there is a need forimproved techniques for the efficient isolation, detection, and accuratequantification of these recently discovered small RNA molecules.

SUMMARY OF THE INVENTION

The present invention concerns methods and compositions for isolating,extracting, purifying, characterizing, quantifying, and/or assayingsmall RNA molecules from a sample, including a cell sample. Suchcompositions and methods allow for manipulation of small RNA molecules,which are often lost or depleted when methods for generally isolatinglarger RNA molecules are employed.

Thus, it is contemplated that the invention concerns small RNAmolecules, which are, in most embodiments, understood to be RNAmolecules of about 100 nucleotides or fewer. Small RNA molecules includesiRNA and miRNA molecules. In some embodiments of the invention, thesmall RNA molecules have at most 100 nucleotides or fewer, have at most70 nucleotides or fewer, or have at most 30 nucleotides or fewer, orhave at most 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15 nucleotides orfewer.

In some cases, the small RNA molecules are double stranded. In somecases, the small RNA molecules are single stranded, though they may haveregions of self-complementarity. There may be 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more such regions, and these regions may involve 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30 or more basepairs (and thus, twice as many bases).Furthermore, these regions of complementarity may involve 100%complementarity or it may involve some mismatches, such as at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity in the regionamong bases, or a region may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 10 ormore mismatches among bases in the region.

It is specifically contemplated that methods and compositions of theinvention can be used to isolate small RNA molecules that are at most100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 24, 23,22, 21, 20, 19, 18, 17, 16, 15, 10 or fewer nucleotides in length, andall ranges derivable between these integers. Furthermore, such moleculescan be isolated so that a sample is enriched in the amount of small RNAmolecules present.

There are several ways in which enrichment and/or purification of smallRNAs may be expressed in the context of the invention. Any increase inthe amount of small RNA molecules present in a sample is within thescope of the invention.

Enrichment and/or purification of small RNAs may be measured in terms ofmass of small RNA relative to mass of total RNA. For example, small RNAin a sample may be enriched about or at least about 1×, 1.5×, 2×, 2.5×,3×, 3.25×, 3.5×, 3.75×, 4×, 4.25×, 4.5×, 4.75×, 5×, 5.25×, 5.5×, 5.75×,6×, 6.25×, 6.5×, 6.75×, 7×, 7.25×, 7.5×, 7.75×, 8×, 8.25×, 8.5×, 8.75×,9×, 9.5×, 10×, 15×, 20×, 25×, 30×, 35×, 40×, 45×, 50×, 55×, 60×, 65×,70×, 75×, 80×, 85×, 90×, 95×, 100×, 110×, 120×, 130×, 140×, 150×, 160×,170×, 180×, 190×, 200×, 210×, 220×, 230×, 240×, 250×, 260×, 270×, 280×,290×, 300×, 325×, 350×, 375×, 400×, 425×, 450×, 475×, 500×, 525×, 550×,575×, 600×, 625×, 650×, 675×, 700×, 725×, 750×, 775×, 800×, 825×, 850×,875×, 900×, 925×, 950×, 975×, 1000×, 1100×, 1200×, 1300×, 1400×, 1500×,1600×, 1700×, 1800×, 1900×, 2000×(same as -fold), and all rangesderivable therein in small RNA molecules as determined by the mass ofsmall RNA molecules relative to the mass of total RNA molecules prior toplacing the lysate on the solid support compared to after eluting theRNA from the solid support.

Enrichment and/or may, alternatively, be measured in terms of the numberof small RNA molecules relative to the number of total RNA molecules.Small RNA molecules can be isolated such that a sample is enriched aboutor at least about 2×, 3×, 4×, 5×, 10×, 15×, 20×, 25×, 30×, 35×, 40×,45×, 50×, 55×, 60×, 65×, 70×, 75×, 80×, 85×, 90×, 95×, 100×, 110×, 120×,130×, 140×, 150×, 160×, 170×, 180×, 190×, 200×, 210×, 220×, 230×, 240×,250×, 260×, 270×, 280×, 290×, 300×, 325×, 350×, 375×, 400×, 425×, 450×,475×, 500×, 525×, 550×, 575×, 600×, 625×, 650×, 675×, 700×, 725×, 750×,775×, 800×, 825×, 850×, 875×, 900×, 925×, 950×, 975×, 1000×, 1100×,1200×, 1300×, 1400×, 1500×, 1600×, 1700×, 1800×, 1900×, 2000×(same as-fold) and all ranges derivable therein in small RNA molecules asdetermined by number of small RNA molecules relative to total number ofRNA molecules prior to placing the lysate on the solid support comparedto after eluting the RNA from the solid support.

Enrichment and/or purification of small RNAs may also be measured interms of the increase of small RNA molecules relative to the number oftotal RNA molecules. Small RNA molecules can be isolated such that theamount of small RNA molecules is increased about or at least about 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% or more with respect to the total amount of RNA in thesample before and after isolation.

Alternatively, in some embodiments, the enrichment and/or purificationof small RNA molecules can be quantified in terms of the absence oflarge RNA molecules present in the sample after eluting the RNA from thesolid support. Small RNA molecules can be enriched such that the numberof RNA molecules larger than 200 nucleotides by mass remaining in thesample after eluting the RNA from the solid support is no more thanabout 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0%, or anyrange therein of the RNA eluted from the solid support.

In some embodiments, about or at least about 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of thesmall RNA molecules in a sample is isolated after the method isimplemented.

Methods of the invention include methods for efficiently isolating smallRNA molecules from cells comprising: a) lysing the cells with a lysingsolution to produce a lysate; b) adding an alcohol solution to thelysate; c) applying the lysate to a solid support; and d) eluting RNAmolecules from the solid support.

Because the small RNA molecules are being efficiently isolated, methodsof the invention include a step of e) using or characterizing the smallRNA molecules. Using or characterizing the small RNA molecules isdistinguished from discarding the small RNA molecules or having them asa byproduct or contaminant in a reaction or assay involving other typesof molecules isolated from the sample, such as longer RNA molecules orDNA molecules.

Samples from which small RNA molecules may be isolated include anysample containing such molecules. The sample may be or contain cells,tissue, organs, or other biological sample. Alternatively, the samplemay be a reaction mixture, such as one in which small RNA molecules wereproduced, generated, or created by enzymatic, synthetic, and/orrecombinant means.

In some embodiments, methods of the invention involve lysing a samplethat contains cells. A “lysate” results when a cell is lysed or itsintegrity disrupted. In specific embodiments of the invention, a lysingsolution is implemented to lyse a cell sample, and the solution includesa chaotropic agent or detergent. A “chaotropic agent” refers to an agentthat unfolds ordered macromolecules, thereby causing them to lose theirfunction (hence causing binding proteins to release their target). A“detergent” refers to a substance that can disperse a hydrophobicsubstance (usually lipids) in water by emulsification. The concentrationof a chaotropic agent in the solutions of the invention, particularlylysing solutions, is about, is at most about, or is at least about 0.5,0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3,3.4, 3.5 M or more, and ranges therein. In specific embodiments, theconcentration of guanidinium in the lysing solution is between about 2.0M and 4.0 M. In some embodiments, the chaotropic agent is guanidiniumchloride or guanidinium isothiocyanate. In still further embodiments,the lysing solution also contains a detergent and/or buffer. Theconcentration of the detergent is between 0.1% to about 2% in someembodiments. The detergent, particularly a mild one that isnondenaturing, can act to solubilize the sample. Detergents may be ionicor nonionic. The ionic detergent N-lauroyl sarcosine is specificallycontemplated for use in solutions of the invention. The concentration ofthe detergent in the buffer may be about, at least about, or at mostabout 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%,5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0% or anyrange therein. It is contemplated that the concentration of thedetergent can be up to an amount where the detergent remains soluble inthe solution.

In other embodiments of the invention, there is a buffer in solutions ofthe invention, including a lysing solution. In specific embodiments, thebuffer is at a concentration of about, at least about, or at most about5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160,165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230,235, 240, 245, 250, 255, 260, 270, 275, 280, 285, 290, 295, 300, 310,320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450,460, 470, 480, 490, 500 mM or any range therein in the solution or inthe solution with the sample. In certain cases, the buffer concentrationin the lysing solution is between about 10 mM and 300 mM. Moreover, inother embodiments, the buffer is TrisCl, although it is contemplatedthat other buffers may be employed as well.

An alcohol solution is added to, mixed with, or incubated with thelysate in embodiments of the invention. An alcohol solution iscontemplated to contain at least one alcohol. The alcohol solution canbe about, be at least about, or be at most about 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% alcohol, orany range therein. In certain embodiments, it is added to a lysate tomake the lysate have a concentration of alcohol of about, about atleast, or about at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,or 90%, or any range therein. In specific embodiments, the amount ofalcohol added to a lysate renders it with an alcohol concentration ofabout 35% to about 70%, or about 50% to about 60%. In specificembodiments, the amount of alcohol solution added to the lysate gives itan alcohol concentration of 55%. Alcohols include, but are not limitedto, ethanol, propanol, isopropanol, and methanol. Ethanol isspecifically contemplated for use in aspects of the invention. It isfurther contemplated that an alcohol solution may be used in additionalsteps of methods of the invention to precipitate RNA.

It is contemplated that the pH of any solution, or of the buffercomponent of any solution, or of any solution with the sample is betweenabout 4.5 and 10.5, though it can be about, about at least, or about atmost 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2 ,5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5,8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9,10.0, 10.1, 10.2, 10.3, 10.4, 10.5 or any range therein.

Other methods of the invention also include extracting small RNAmolecules from the lysate with an extraction solution comprising anon-alcohol organic solvent prior to applying the lysate to the solidsupport. In specific embodiments, the extraction solution contains anon-alcohol organic solvent such as phenol and/or chloroform. Thenon-alcohol organic solvent solution is understood to contain at leastone non-alcohol organic solvent, though it may also contain an alcohol.The concentrations described above with respect to alcohol solutions areapplicable to concentrations of solutions having non-alcohol organicsolvents. In specific embodiments, equal amounts of 1) the lysate and 2)phenol and/or chloroform are mixed. In specific embodiments, the alcoholsolution is added to the lysate before extraction with a non-alcoholorganic solvent.

Extraction of RNA from the lysate includes using a solid support, suchas a mineral or polymer support. A “solid support” refers to a physicalstructure containing a material which contacts the lysate and that doesnot irreversibly react to macromolecules in the lysate, particularlywith small RNA molecules In particular embodiments, the solid supportbinds small RNA molecules; in additional cases, it binds small RNAmolecules, but does not bind one or more other types of macromoleculesin the sample. The material in the solid support may include a mineralor polymer, in which case the support is referred to as a “mineral orpolymer support.” Mineral or polymer supports include supports involvingsilica. In some embodiments, the silica is glass. Supports include, butare not limited to, beads, columns and filters. In further embodiments,the mineral or polymer support is a glass fiber filter or column.

Alternatively, in some embodiments, the mineral or polymer support mayinclude polymers or nonpolymers with electronegative groups. In someembodiments, the material is or has polyacrylate, polystyrene, latex,polyacrylonitrile, polyvinylchloride, methacrylate, and/or methylmethacrylate. Such supports are specifically contemplated for use withthe present invention.

In some methods of the invention, a lysate that may or may not have beenmixed with an alcohol or non-alcohol organic solvent solution is appliedto a solid support and the RNA is eluted from the support.

After a lysate is applied or mixed with a solid support, the materialmay be washed with a solution. In some embodiments, a mineral or polymersupport is washed with a first wash solution after applying the lysateto the mineral or polymer support. In further embodiments, a washsolution comprises a chaotropic or reducing agent. The chaotropic agentis guanidinium in some wash solutions. A wash solution includes alcoholin some embodiments of the invention, and in some cases, it has bothalcohol and guanidinium. It is further contemplated that methods of theinvention involve 1, 2, 3, 4, 5 or more washes with a wash solution. Thewash solution used when more than one washing is involved may be thesame or different. In some embodiments, the wash solutions have the samecomponents but in different concentrations from each other. It isgenerally understood that molecules that come through the material in awash cycle are discarded.

In other methods of the invention, the desired RNA molecules are elutedfrom the solid support. In certain embodiments, small RNA molecules areeluted from a solid support such as a mineral or polymer support at atemperature of about 60° C. to about 100° C. It is contemplated that thetemperature at which the RNA molecules are eluted is about or at leastabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100° C. or more, or any range therein. The molecules may beeluted with any elution solution. In some embodiments, the elutionsolution is an ionic solution, that is, it includes ions. In particularembodiments, the elution solution includes up to 10 mM salt. It iscontemplated to be about, at least about, or at most about 0.1, 0.5, 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more mM salt. In certain embodiments, thesalt consists of a combination of Li⁺,Na⁺, K⁺, or NH₄ ⁺ as cation andCl⁻, Br⁻, I⁻, ethylenediaminetetraacetate, or citrate as anion.

Additional method steps include passing the small RNA molecules througha GFF while binding only the larger RNAs. In some embodiments, thepassed small RNA molecules are captured on a second GFF and then eluted.Material that is not captured on the second GFF filter is discarded ornot used in additional methods of the invention.

Specific methods of the invention concern isolating miRNA or siRNA froma sample by at least the following steps: a) obtaining a sample havingmiRNA or siRNA; b)adding an extraction solution to the sample; c) addingan alcohol solution to the extracted sample; d) applying the sample to amineral or polymer support; and, e) eluting the RNA containing siRNA ormiRNA from the mineral or polymer support with an ionic solution. Inparticular embodiments, the sample is a cell lysate. The cell lysate, insome cases, is produced by adding a lysing solution comprising achaotropic agent or detergent to cells having miRNA or siRNA. In someembodiments, the eluted sample is enriched at least about 10-fold formiRNA and/or siRNA by mass.

Additional methods for isolating miRNA molecules from a sample involve:a) adding an alcohol solution to the sample; b) applying the sample to amineral or polymer solid support; c) eluting miRNA molecules from thesupport with an ionic solution; and, d) using or characterizing themiRNA molecules.

Other methods for isolating small RNA molecules from a sample include:a) lysing cells in the sample with a lysing solution comprisingguanidinium, wherein a lysate with a concentration of at least about 1 Mguanidinium is produced; b) extracting small RNA molecules from thelysate with an extraction solution comprising phenol; c) adding to thelysate an alcohol solution for form a lysate/alcohol mixture, whereinthe concentration of alcohol in the mixture is between about 35% toabout 70%; d) applying the lysate/alcohol mixture to a solid support; e)eluting the small RNA molecules from the solid support with an ionicsolution; f) capturing the small RNA molecules; and, g) using theisolated small RNA molecules.

After RNA is extracted, individual or specific RNA molecules and/orpools of RNA molecules (as well as the entire population of isolatedRNA) can be subject to additional reactions and/or assays. In somecases, these reactions and/or assays involve amplification of the RNA orof a DNA molecule generated from the RNA. For example, RT-PCR may beemployed to generate molecules that can be characterized.

In some embodiments, a particular RNA molecule or an RNA population maybe quantified or characterized. Quantification includes any procedureknown to those of skill in the art such as those involving one or moreamplification reactions or nuclease protection assays, such as thoseusing ribonuclease to discriminate between probe that is hybridized to aspecific miRNA target or unhybridized, as embodied in the mirVana miRNADetection Kit from Ambion. These procedures include quantitative reversetranscriptase-PCR (qRT-PCR). In some embodiments, characterization ofthe isolated RNA is performed. cDNA molecules are generated from theextracted RNA. Other characterization and quantification assays arecontemplated as part of the invention. The methods and compositions ofthe invention allow small RNA molecules to be quantified andcharacterized. The small RNA molecules can also be used with arrays; togenerate cDNAs for use in arrays or as targets to be detected by arrays,after being labeled by radioactive, fluorescent, or luminescent tags.Other assays include the use of spectrophotometry, electrophoresis, andsequencing.

The present invention also concerns kits for isolating small RNAmolecules, such as miRNA and/or siRNA from a sample, particularly a cellsample. Thus, any of the compositions discussed above can be includedwith any other composition discussed above for inclusion in a kit. Insome embodiments, there are kits for isolating small RNA moleculescomprising: a) acid phenol-chloroform; b) a lysis/binding buffer, c) asmall RNA homogenate additive, d) one or more small RNA washsolution(s), and e) an elution solution.

In preferred embodiment, the kit contains: a) an acid phenol-chloroform;b) a lysis/binding buffer comprising 4 M GuSCN, 0.1 Mbeta-mercaptoethanol, 0.5% N-lauroyl sarcosine, 25 mM Na-citrate, pH7.2; c) a small RNA homogenate additive comprising 2 M sodium acetate,pH 4, to be added in 0.1 volume before extraction with PC; d) a washsolution #1 comprising 1.6 M GuSCN in 70% ethanol; e) a wash solution#2/3 comprising 80% ethanol, 0.1 M NaCl, 4.5 mM EDTA, 10 mM TrisHCl, pH7.5; f) an elution solution comprising 0.1 mM EDTA, pH8; g) a gelloading buffer II; h) collection tubes; and i) filter cartridges.

In some embodiments, kits of the invention include one or more of thefollowing in a suitable container means (consistent with compositionsdiscussed above): a lysis buffer with a chaotropic agent; a glass fiberfilter or column; elution buffer; wash buffer; alcohol solution; andRNase inhibitor.

The components of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits will generally includeat least one vial, test tube, flask, bottle, syringe or other containermeans, into which a component may be placed, and preferably, suitablyaliquoted. Where there are more than one component in the kit (they maybe packaged together), the kit also will generally contain a second,third or other additional container into which the additional componentsmay be separately placed. However, various combinations of componentsmay be comprised in a vial. The kits of the present invention also willtypically include a means for containing the RNA, and any other reagentcontainers in close confinement for commercial sale. Such containers mayinclude injection or blow-molded plastic containers into which thedesired vials are retained. When the components of the kit are providedin one and/or more liquid solutions, the liquid solution is an aqueoussolution, with a sterile aqueous solution being particularly preferred.

However, the components of the kit may be provided as dried powder(s).When reagents and/or components are provided as a dry powder, the powdercan be reconstituted by the addition of a suitable solvent. It isenvisioned that the solvent may also be provided in another containermeans. The container means will generally include at least one vial,test tube, flask, bottle, syringe and/or other container means, intowhich the nucleic acid formulations are placed, preferably, suitablyallocated. The kits may also comprise a second container means forcontaining a sterile, pharmaceutically acceptable buffer and/or otherdiluent.

Such kits may also include components that preserve or maintain the RNAor that protect against its degradation. Such components may beRNAse-free or protect against

RNAses. Such kits generally will comprise, in suitable means, distinctcontainers for each individual reagent or solution.

It is contemplated that any embodiment discussed herein can beimplemented with respect to any method or composition of the invention,and vice versa. Furthermore, compositions and kits of the invention canbe used to achieve methods of the invention.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

Following long-standing patent law, the words “a” and “an,” when used inconjunction with the word “comprising” in the claims or specification,denotes one or more, unless specifically noted.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Binding behavior of let-7 miRNA in raw extracts from mousebrain, heart, and liver at various ethanol concentrations. Absoluteethanol was added to crude lysate to create the final ethanolconcentrations indicated.

FIG. 2. Binding behavior of let-7 miRNA in phenol-chloroform extractsfrom mouse brain, heart, and liver at various ethanol concentrations.Absolute ethanol was added to lysates after extraction byphenol-chloroform (as in the standard procedure) to create the finalethanol concentrations indicated.

FIG. 3. Binding behavior of β-actin, GAPDH, PPI, U2, and let-7 atvarying ethanol concentrations in the presence of 2M GuSCN, with ethanolconcentration ajusted by addition of an equal volume of a 2× ethanolsolution in water.

FIG. 4. Binding behavior of β-actin, GAPDH, PPI, U2, and let-7 atvarying ethanol concentrations in the presence of 2M GuSCN, with ethanolconcentration adjusted by addition of absolute ethanol.

FIG. 5. Binding behavior of β-actin, GAPDH, PPI, U2, and let-7 atvarying ethanol concentrations in the presence of 3M GuSCN, with ethanolconcentration adjusted by addition of an equal volume of a 2× ethanolsolution in water.

FIG. 6. Binding behavior of β-actin, GAPDH, PPI, U2, and let-7 atvarying ethanol concentrations in the presence of 3M GuSCN, with ethanolconcentration adjusted by addition of absolute ethanol.

FIG. 7. Relative enrichment of β-actin, GAPDH, U2, and let-7 RNAs.

FIG. 8. Relative enrichment of U2 and let 7 RNAs.

FIG. 9. Yield of current procedure compared to standardphenol-chloroform extraction and ethanol precipitation.

FIG. 10. Comparison of absolute yield of three small RNAs, let-7 (22nt), U43 (62 nt), and U2 (187 nt), using the current process (AmbionmicroRNA Isolation Kit=AMIK) versus a glass fiber system currentlyavailable (RNeasy).

FIG. 11. Comparison of yield from cultured cells, with and withoutpre-extraction. The yield of both U2 and let-7 were determined.

FIG. 12. Effects of different concentrations of NaOOCH₃ at two pHs forthe phenol-chloroform extraction prior to glass immobilization on yieldof U2, U43, and let-7.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. Small RNA Molecules

Natural populations of RNA are routinely isolated from animal and planttissue as well as cells grown in culture. However, most of theseprocedures are unconcerned with retaining small RNAs, in the range ofless than 100 nucleotides long. In fact, it is known that standardprecipitation procedures with alcohol are inefficient in capturingnucleic acids smaller than around 100 nucleotides.

The presence of small RNA molecules and free nucleotides has long beenobserved in RNA extracted from biological samples and assumed to reflectthe breakdown products of larger protein-coding and functional RNAs,including those involved in translation and RNA processing complexes. Inthe past few years, small RNAs involved in the regulation of geneexpression have been found to be present in virtually all eukaryoticorganisms. In 1993, the Ambrose lab published a report on the discoverythat the let-7 gene, which results in developmental mis-timing, orheterochromy, in the nematode Caenorhabditis elegans coded for a 22-ntRNA (Lee et al., 1993.). This small, single-stranded RNA (now termedmicroRNA or miRNA) affects the expression of a set of developmentalgenes by inhibiting their ability to function in translation based onpartial sequence complementarity with the targeted gene. The presence ofthis small RNA was found to be conserved in many evolutionarilydivergent species (Pasquinelli et al., 2000), including vertebrate,ascidian, hemichordate, mollusc, annelid and arthropod.

In 2001, several groups used a novel cloning method to isolate andidentify a large variety of these “micro RNAs” (miRNAs) from C. elegans,Drosophila, and humans (Lagos-Quintana et al., 2001; Lau et al., 2001;Lee and Ambros, 2001). Several hundreds of miRNAs have been identifiedin plants and animals.

miRNAs thus far observed have been approximately 21-22 nucleotides inlength and they arise from longer precursors, which are transcribed fromnon-protein-encoding genes. See review of Carrington et al. (2003). Theprecursors form structures that fold back on each other inself-complementary regions; they are then processed by the nucleaseDicer in animals or DCL1 in plants. miRNA molecules interrupttranslation through imprecise base-pairing with their targets.

Micro RNAs are not the only RNAs of that size found in eukaryotic cells.A pathway for degradation of mRNAs in the cell was found that createssmall double-stranded RNAs (Fire et al., 1998; Zamore et al., 2000; manyothers, reviewed in Timmons, 2002.). This process, called RNAinterference, uses these “small interfering RNAs” (siRNAs) to targettheir degradation sites, usually from a much larger double-strandedintermediate. Although the natural function of this system is not known,it is thought to be involved in the response to infective agents. Plantshave been found to have a similar system, which also utilizes microRNAsin post-transcriptional gene-silencing (Hamilton and Baulcombe, 1999;Tang et al. 2003).

II. Isolation of Short RNA Molecules

Methods of the invention involve one or more steps to efficientlyisolate and/or enrich short RNA molecules. These steps include orinvolve the following: lysing cells and/or creating a cell lysate;

A. Creating Cell Lysates

It is contemplated that the present invention can be used to facilitatepreparation of small RNA molecules from biological samples forevaluation and subsequent use. In some embodiments of the invention,preparation of samples involves homogenizing the sample or preparing acell lysate from the sample. In embodiments of the invention,homogenization or lysing of a cell is accomplished using a solution thatcontains a guanidinium salt, detergent, surfactant, or other denaturant.The terms homogenization and lysing are used interchangeably.

Guanidinium salts are well known to those of skill in the art andinclude guanidinium hydrochloride and guanidinium isothiocyanate. Insome embodiments, they may be present in a concentration of about 2 toabout 5 M. Additionally, a homogenization solution may contain urea orother denaturants such as NaI.

In embodiments of the invention, a buffer is included in the lysis orhomogenization solution. In certain cases, the buffer is TrisCl.

A biological sample may be homogenized or fractionated in the presenceof a detergent or surfactant. The detergent can act to solublize thesample. Detergents may be ionic or nonionic. Examples of nonionicdetergents include triton, such as the Triton X series (Triton X-100,Triton X-100R, Triton X-114, Triton X-450, Triton X-450R), octylglucoside, polyoxyethylene(9)dodecyl ether, digitonin, IGEPAL CA630,n-octyl-beta-D-glucopyranoside (betaOG), n-dodecyl-beta, C12EO7, Tween20, Tween 80, polidocanol, n-dodecyl beta-D-maltoside (DDM), NP-40,C12E8 (octaethylene glycol n-dodecyl monoether), hexaethyleneglycolmono-n-tetradecyl ether (C14EO6), octyl-beta-thioglucopyranoside (octylthioglucoside, OTG), Emulgen, and polyoxyethylene 10 lauryl ether(C12E10). Examples of ionic detergents (anionic or cationic) includedeoxycholate, sodium dodecyl sulfate (SDS), N-lauroylsarcosine, andcetyltrimethylammoniumbromide (CTAB). A zwitterionic reagent may also beused in the purification schemes of the present invention, such asChaps, zwitterion 3-14, and3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. It iscontemplated also that urea may be added with or without anotherdetergent or surfactant.

Lysis or homogenization solutions may further contain other agents suchas reducing agents. Examples of such reducing agents includedithiothreitol (DTT), β-mercaptoethanol, DTE, GSH, cysteine, cysteamine,tricarboxyethyl phosphine (TCEP), or salts of sulfurous acid.

In some embodiments of the invention, a lysis solution includes:guanidinium thiocyanate, N-lauroyl sarcosine, and TrisHCl. Once thesample has been homogenized into this solution, the RNA can beextracted, often with phenol solutions or the use of an adsorptive solidphase. Alternative methods use combination denaturant/phenol solutionsto perform the initial homogenization, precluding the need for asecondary extraction. Examples of these reagents would be Trizol™(Invitrogen) or RNAwiz™ (Ambion, Inc.)

Subsequent to exposure to a homogenization solution, samples may befurther homogenized by mechanical means. Mechanical blenders,rotor-stator homogenizers, or shear-type homogenizers may be employed.

Alternatively, the tissue could be homogenized in the lysis solution,and the tissue remains separated by settling, centrifugation, orfiltration. These remains could then be treated with homogenizationsolution and extraction conditions as described above.

The methods of the invention may further include steps involvingremoving lipids or compositions thereof with detergents or surfactants.A lipid may be naturally occurring or synthetic (i.e., designed orproduced by man). However, a lipid is usually a biological substance.Biological lipids are well known in the art, and include for example,neutral fats, phospholipids, phosphoglycerides, steroids, terpenes,lysolipids, glycosphingolipids, glucolipids, sulphatides, lipids withether and ester-linked fatty acids and polymerizable lipids, andcombinations thereof. Removal of a lipid such as a phospholipid isdescribed herein.

Detergents may be used to facilitate homogenization or the creation of acell lysate. These detergents specifically include Triton X-100 andCHAPS. CHAPS is the zwitterionic detergent3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate.

B. Extracting Small RNA Molecules

After lysing or homogenizing a cell sample, additional procedures may beimplemented to extract specifically RNA molecules. It is contemplatedthat if the sample involves cells, the step of lysing or homogenizingcan be considered part of an overall extraction process, however, theextraction of RNA specifically may be referred to, and will beunderstood as separating RNA molecules from other biomolecules such aslipids and proteins. Extraction of RNA molecules from these otherstructures can involve extraction solutions containing one or moreorganic solvents. In some cases, the organic solvent is a non-alcoholorganic solvent such as phenol and/or chloroform. In others, a solutioncontains an alcohol, which may be any alcohol used for the extraction ofnucleic acids, but in certain embodiments, the alcohol is ethanol.

RNA molecules may be extracted from a variety of cell samples. Such cellsamples may comprise cells of the brain, head, neck, gastrointestinaltract, lung, liver, pancreas, breast, testis, uterus, bladder, kidney,prostate, colon, kidney, skin, ovary, and heart but is not limited tosuch cells.

The term “nucleic acid” is well known in the art. A “nucleic acid” asused herein will generally refer to a molecule (i.e., a strand) of DNA,RNA or a derivative or analog thereof, comprising a nucleobase. Anucleobase includes, for example, a naturally occurring purine orpyrimidine base found in DNA (e.g., an adenine “A,” a guanine “G,” athymine “T” or a cytosine “C”) or RNA (e.g., an A, a G, an uracil “U” ora C). The term “nucleic acid” or “RNA molecule” encompasses the terms“oligonucleotide” and “polynucleotide,” each as a subgenus of the term“nucleic acid.”

A nucleic acid “complement(s)” or is “complementary” to another nucleicacid when it is capable of base-pairing with another nucleic acidaccording to the standard Watson-Crick, Hoogsteen or reverse Hoogsteenbinding complementarity rules. As used herein “another nucleic acid” mayrefer to a separate molecule or a spatial separated sequence of the samemolecule.

As used herein, the term “complementary” or “complement(s)” also refersto a nucleic acid comprising a sequence of consecutive nucleobases orsemi-consecutive nucleobases (e.g., one or more nucleobase moieties arenot present in the molecule) capable of hybridizing to another nucleicacid strand or duplex even if less than all the nucleobases do not basepair with a counterpart nucleobase. In certain embodiments, a“complementary” nucleic acid comprises a sequence in which about 70%,about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about77%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%,about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, about 99%, to about 100%, and any rangederivable therein, of the nucleobase sequence is capable of base-pairingwith a single or double stranded nucleic acid molecule duringhybridization. In certain embodiments, the term “complementary” refersto a nucleic acid that may hybridize to another nucleic acid strand orduplex in stringent conditions, as would be understood by one ofordinary skill in the art.

In certain embodiments, a “partly complementary” nucleic acid comprisesa sequence that may hybridize in low stringency conditions to a singleor double stranded nucleic acid, or contains a sequence in which lessthan about 70% of the nucleobase sequence is capable of base-pairingwith a single or double stranded nucleic acid molecule duringhybridization.

These definitions generally refer to a single-stranded molecule, but inspecific embodiments will also encompass an additional strand that ispartially, substantially or fully complementary to the single-strandedmolecule. Thus, a nucleic acid may encompass a double-stranded moleculeor a triple-stranded molecule that comprises one or more complementarystrand(s) or “complement(s)” of a particular sequence comprising amolecule. As used herein, a single stranded nucleic acid may be denotedby the prefix “ss,” a double stranded nucleic acid by the prefix “ds,”and a triple stranded nucleic acid by the prefix “ts.”

C. Solid Support and Devices

A solid support is a structure containing material that will reversiblybind with nucleic acids, particularly small RNA molecules, and in someembodiments, it will not bind one or more other types of macromoleculesin the sample. Material may comprise plastic, glass, silica, a magnet, ametal such as gold, carbon, cellulose, latex, polystyrene, and othersynthetic polymers, nylon, cellulose, nitrocellulose, polyacrylate,polyacrylonitrile, methacrylate, and/or methyl methacrylatepolymethacrylate, polyvinylchloride, styrene-divinylbenzene, or anychemically-modified plastic. They may also be porous or non-porousmaterials. The structure may also be a particle of any shape that allowsthe small RNA molecules to be isolated, depleted, or separated. In someembodiments, it is a column that includes any of the materials describedabove through which a lysate may be passed.

Other components include isolation apparatuses such as filtrationdevices, including spin filters or spin columns. It may be a sphere,such as a bead, or a rod, or a flat-shaped structure, such as a platewith wells. The structure and sample containing the desired RNAmolecules may be centrifuged, filtered, dialyzed, and/or otherwiseisolated. When the structure is centrifuged it may be pelleted or passedthrough a centrifugible filter apparatus.

The structure may also go through an additional capture step. In someembodiments, the sample is subsequently filtered after passage through acapture structure. The capture step can include filtration using apressure-driven system or gravity-based system (for example,centrifugation). Many such structures are available commercially and maybe utilized herewith. Other examples can be found in WO 86/05815,WO90/06045, U.S. Pat. 5,945,525, all of which are specificallyincorporated by reference.

II. Characterization and Quantitation of Isolated Small RNA Molecules

Small RNA molecules obtained from samples may be analyzed or quantitatedby various methods to characterize them, quantitate them, or use themfor analysis of other biological samples. Provided herein are methods ofquantitating or analyzing RNA, or manipulating the RNA for use in assaysinvolving other biological material. General methods for quantitating oranalyzing RNA may be found in Sambrook et al. (2001) or Maniatis et al.(1990). Below are provides examples of for using small RNA moleculesfrom samples, however, these examples and are not meant to be limiting.

A. Nuclease Protection Assays

Nuclease protection assays (NPAs), including both ribonucleaseprotection assays (RPAs) and 51 nuclease assays, are an extremelysensitive method for the detection, quantitation and mapping of specificRNAs in a complex mixture of total cellular RNA. The basis of NPAs is asolution hybridization of a single-stranded, discrete sized antisenseprobe(s) to an RNA sample. The small volume solution hybridization isfar more efficient than more common membrane-based hybridization, andcan accommodate up to 100 μg of total or poly(A) RNA. Afterhybridization, any remaining unhybridized probe and sample RNA areremoved by digestion with a mixture of nucleases. Then, in a single stepreaction, the nucleases are inactivated and the remaining probe:targethybrids are precipitated. These products are separated on a denaturingpolyacrylamide gel and are visualized by autoradiography. If nonisotopicprobes are used, samples are visualized by transferring the gel to amembrane and performing secondary detection.

Such techniques are well known to those of ordinary skill in the art.Commercial kits for such assays are readily available, such as theDirect Protect™ Lysate RPA Kit, HybSpeed™ RPA Kit, and RPA II and RPAIII™ Ribonuclease Protection Assay Kits from Ambion.

B. Denaturing Agarose Gel Electrophoresis

Small RNA molecules isolated from a sample may be quantitated by gelelectrophoresis using a denaturing gel system. Acrylamide gels are thepreferred matrix for separations of this size, although highconcentrations (˜4%+) of modified agarose such as NuSieve (FMC, 191Thomaston St., Rockland, Me. 04841) can also be used. A positive controlshould be included on the gel so that any unusual results can beattributed to a problem with the gel or a problem with the RNA underanalysis. RNA molecular weight markers, an RNA sample known to beintact, or both, can be used for this purpose. It is also a good idea toinclude a sample of the starting RNA that was used in the enrichmentprocedure. The presence of specific small RNAs can be determined byblotting the contents of these gels onto hybridization membranes andprobing with radioactive oligonucleotide

(RNA or DNA-based) probes.

C. Assessing RNA Yield by UV Absorbence The concentration and purity ofRNA can be determined by diluting an aliquot of the preparation (usuallya 1:50 to 1:100 dilution) in TE (10 mM Tris-HCl pH 8, 1 mM EDTA) orwater, and reading the absorbence in a spectrophotometer at 260 nm and280 nm.

An A₂₆₀ of 1 is equivalent to 40 μg RNA/ml. The concentration (μg/ml) ofRNA is therefore calculated by multiplying the A₂₆₀ X dilution factor X40 μg/ml. The following is a typical example:

The typical yield from 10 μg total RNA is 3-5 μg. If the sample isre-suspended in 25 μl, this means that the concentration will varybetween 120 ng/μl and 200 ng/μl. One μl of the prep is diluted 1:50 into49 μl of TE. The A₂₆₀=0.1. RNA concentration=0.1×50×40 μg/ml=200 μg/mlor 0.2 μg/μl. Since there are 24 μl of the prep remaining after using 1μl to measure the concentration, the total amount of remaining RNA is 24μl=0.2 μg/μl=4.8 μg.

D. Other Uses of Small RNA Molecules from Samples

Small RNA molecules obtained from a sample may be analyzed by or used inmicroarray technology. For example an arrays such as a gene array aresolid supports upon which a collection of gene-specific probes has beenspotted at defined locations. The probes localize complementary labeledtargets from a nucleic acid sample, such as an RNA sample, populationvia hybridization. One of the most common uses for gene arrays is thecomparison of the global expression patterns of an RNA population.Typically, RNA isolated from two or more tissue samples may be used. TheRNAs are reverse transcribed using labeled nucleotides and targetspecific, oligodT, or random-sequence primers to create labeled cDNApopulations. The cDNAs are denatured from the template RNA andhybridized to identical arrays. The hybridized signal on each array isdetected and quantified. The signal emitting from each gene-specificspot is compared between the populations. Genes expressed at differentlevels in the samples generate different amounts of labeled cDNA andthis results in spots on the array with different amounts of signal.

The direct conversion of RNA populations to labeled cDNAs is widely usedbecause it is simple and largely unaffected by enzymatic bias. However,direct labeling requires large quantities of RNA to create enoughlabeled product for moderately rare targets to be detected by arrayanalysis. Most array protocols recommend that 2.5 g of polyA or 50 g oftotal RNA be used for reverse transcription (Duggan, 1999). Forpractitioners unable to isolate this much RNA from their samples, globalamplification procedures have been used.

The most often cited of these global amplification schemes is antisenseRNA (aRNA) amplification (U.S. Pat. Nos. 5,514,545 and 5,545,522).Antisense RNA amplification involves reverse transcribing RNA sampleswith an oligo-dT primer that has a transcription promoter such as the T7RNA polymerase consensus promoter sequence at its 5′ end. First strandreverse transcription creates single-stranded cDNA. Following firststrand cDNA synthesis, the template RNA that is hybridized to the cDNAis partially degraded creating RNA primers. The RNA primers are thenextended to create double-stranded DNAs possessing transcriptionpromoters. The population is transcribed with an appropriate RNApolymerase to create an RNA population possessing sequence from thecDNA. Because transcription results in tens to thousands of RNAs beingcreated from each DNA template, substantive amplification can beachieved. The RNAs can be labeled during transcription and used directlyfor array analysis, or unlabeled aRNA can be reverse transcribed withlabeled dNTPs to create a cDNA population for array hybridization. Ineither case, the detection and analysis of labeled targets are wellknown in the art. Other methods of amplification that may be employedinclude, but are not limited to, polymerase chain reaction (referred toas PCR™; see U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, andInnis et al., 1988); and ligase chain reaction (“LCR”), disclosed inEuropean Application No. 320 308, U.S. Pat. Nos. 4,883,750, 5,912,148.Qbeta Replicase, described in PCT Application No. PCT/US87/00880, mayalso be used as an amplification method Alternative methods foramplification of a nucleic acid such as RNA are disclosed in U.S. Pat.Nos. 5,843,650, 5,846,709, 5,846,783, 5,849,546, 5,849,497, 5,849,547,5,858,652, 5,866,366, 5,916,776, 5,922,574, 5,928,905, 5,928,906,5,932,451, 5,935,825, 5,939,291, 5,916,779 and 5,942,391, GB ApplicationNo. 2 202 328, and in PCT Application No. PCT/US89/01025, PCTApplication WO 89/06700, PCT Application WO 88/10315, EuropeanApplication No. 329 822, Kwoh et al., 1989; Frohman, 1994; Ohara et al.,1989; and Walker et al., 1992 each of which is incorporated herein byreference in its entirety.

cDNA libraries may also be constructed and used to analyze to the RNAextracted from a sample. Construction of such libraries and analysis ofRNA using such libraries may be found in Sambrook et al. (2001);Maniatis et al. (1990); Efstratiadis et al. (1976); Higuchi et al.(1976); Maniatis et al. (1976); Land et al. (1981); Okayama et al.(1982); Gubler et al. (1983); Ko (1990); Patanjali et al. (1991); U.S.Patent Appin. 20030104468, each incorporated herein by reference.

The present methods and kits may be employed for high volume screening.A library of RNA or DNA can be created using methods and compositions ofthe invention. This library may then be used in high throughput assays,including microarrays. Specifically contemplated by the presentinventors are chip-based nucleic acid technologies such as thosedescribed by Hacia et al. (1996) and Shoemaker et al. (1996). Briefly,these techniques involve quantitative methods for analyzing largenumbers of genes rapidly and accurately. By using fixed probe arrays,one can employ chip technology to segregate target molecules as highdensity arrays and screen these molecules on the basis of hybridization(see also, Pease et al., 1994; and Fodor et al, 1991). The term “array”as used herein refers to a systematic arrangement of nucleic acid. Forexample, a nucleic acid population that is representative of a desiredsource (e.g., human adult brain) is divided up into the minimum numberof pools in which a desired screening procedure can be utilized todetect or deplete a target gene and which can be distributed into asingle multi-well plate. Arrays may be of an aqueous suspension of anucleic acid population obtainable from a desired mRNA source,comprising: a multi-well plate containing a plurality of individualwells, each individual well containing an aqueous suspension of adifferent content of a nucleic acid population. Examples of arrays,their uses, and implementation of them can be found in U.S. Pat. Nos.6,329,209, 6,329,140, 6,324,479, 6,322,971, 6,316,193, 6,309,823,5,412,087, 5,445,934, and 5,744,305, which are herein incorporated byreference.

Microarrays are known in the art and consist of a surface to whichprobes that correspond in sequence to gene products (e.g., cDNAs, mRNAs,cRNAs, polypeptides, and fragments thereof), can be specificallyhybridized or bound at a known position. In one embodiment, themicroarray is an array (i.e., a matrix) in which each positionrepresents a discrete binding site for a product encoded by a gene(e.g., a protein or RNA), and in which binding sites are present forproducts of most or almost all of the genes in the organism's genome. Ina preferred embodiment, the “binding site” (hereinafter, “site”) is anucleic acid or nucleic acid analogue to which a particular cognate cDNAcan specifically hybridize. The nucleic acid or analogue of the bindingsite can be, e.g., a synthetic oligomer, a full-length cDNA, a less-thanfull length cDNA, or a gene fragment.

The nucleic acid or analogue are attached to a solid support, which maybe made from glass, plastic (e.g., polypropylene, nylon),polyacrylamide, nitrocellulose, or other materials. A preferred methodfor attaching the nucleic acids to a surface is by printing on glassplates, as is described generally by Schena et al., 1995a. See alsoDeRisi et al., 1996; Shalon et al., 1996. Other methods for makingmicroarrays, e.g., by masking (Maskos et al., 1992), may also be used.In principal, any type of array, for example, dot blots on a nylonhybridization membrane (see Sambrook et al., 2001, which is incorporatedin its entirety for all purposes), could be used, although, as will berecognized by those of skill in the art, very small arrays will bepreferred because hybridization volumes will be smaller.

Use of a biochip is also contemplated, which involves the hybridizationof a labeled molecule or pool of molecules to the targets immobilized onthe biochip.

III. Kits In further embodiments of the invention, there is a provided akit for the isolation of small RNA molecules, such as miRNA and siRNAfrom a sample, particularly a cell sample. Any of the compositionsdescribed herein may be comprised in a kit. In a non-limiting example,reagents for lysing cells, extracting RNA the cell lysate, and/oranalyzing or quantitating the RNA obtained may be included in a kit. Thekits will thus comprise, in suitable container means, any of thereagents disclosed herein. It may also include one or more buffers orsolutions, such as lysis buffer, extraction buffer, solutions to havealcohol added, elution solution, wash solution and other components forisolating the desired RNA, such as a capture structure. In someembodiments, there are kits for isolating small RNA moleculescomprising: a) Acid Phenol-Chloroform; b) Lysis/Binding Buffer(GuSCN-based); c) miRNA Homogenate Additive (2M Sodium Acetate, pH 4, tobe added in 0.1 vol before extraction with PC); d) miRNA Wash Soln #1(1.6M GuSCN in 70% ethanol); e) Wash Soln #2/3 (80% ethanol, 0.1 M NaCl,4.5 mM EDTA, 10 mM TrisHCl, pH 7.5); f) Elution Solution (0.1 mM EDTA,pH8); g) Gel Loading Buffer II; h) Collection Tubes; i) FilterCartridges.

The components of the kits may be packaged either in aqueous media or inlyophilized form. The container means of the kits will generally includeat least one vial, test tube, flask, bottle, syringe or other containermeans, into which a component may be placed, and preferably, suitablyaliquoted. Where there are more than one component in the kit (they maybe packaged together), the kit also will generally contain a second,third or other additional container into which the additional componentsmay be separately placed. However, various combinations of componentsmay be comprised in a vial. The kits of the present invention also willtypically include a means for containing the RNA, and any other reagentcontainers in close confinement for commercial sale. Such containers mayinclude injection or blow-molded plastic containers into which thedesired vials are retained. When the components of the kit are providedin one and/or more liquid solutions, the liquid solution is an aqueoussolution, with a sterile aqueous solution being particularly preferred.

However, the components of the kit may be provided as dried powder(s).When reagents and/or components are provided as a dry powder, the powdercan be reconstituted by the addition of a suitable solvent. It isenvisioned that the solvent may also be provided in another containermeans. The container means will generally include at least one vial,test tube, flask, bottle, syringe and/or other container means, intowhich the nucleic acid formulations are placed, preferably, suitablyallocated. The kits may also comprise a second container means forcontaining a sterile, pharmaceutically acceptable buffer and/or otherdiluent.

Such kits may also include components that preserve or maintain the RNAor that protect against its degradation. Such components may beRNAse-free or protect against RNAses. Such kits generally will comprise,in suitable means, distinct containers for each individual reagent orsolution.

A kit will also include instructions for employing the kit components aswell the use of any other reagent not included in the kit. Instructionsmay include variations that can be implemented.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Preparation of Cell Lysate and Isolation of RNA

The following procedure provides the basis for the invention and isreferred to in the Examples as the Ambion miRNA Isolation Kit (AMIK)procedure.

Frozen tissue was ground under liquid nitrogen to a fine powder. Lysisbuffer (4 M GuSCN; 0.1 M beta-mercaptoethanol; 0.5% N-lauroyl sarcosine;25 mM Na-citrate, pH 7.2) was added to this powder in an appropriatevessel at a proportion of 1 ml to every gram of tissue powder. This washomogenized using mechanical means to create a finely-dispersed tissuelysate. One tenth volume of a 2 M Na acetate (pH 4.0) solution was addedand mixed thoroughly, adding 0.1 ml for every ml. The lysate was thenprocessed immediately (without organic extraction) or placed on ice tobe processed within 15 minutes.

Processing involved the addition of an equal volume of AcidPhenol-Chloroform (5:1, equilibrated with aqueous solution at pH 4.5) tothe suspension, followed by vigorous agitation (by vortexing or shaking)for 30-60 sec. The phenol-chloroform and aqueous phases were thenseparated by centrifugation at 16,000 ×G for 5 min, or until a clearinterface was obtained. The aqueous phase was removed by aspiration,avoiding withdrawing any of the interface between phases. This aqueousphase, which contained the RNA from the sample, was made into aconcentration of 55% ethanol by addition of 1.22 volumes of ethanol.

Immediately after mixing, the sample was applied directly to a glassfiber column, as used in an RNAqueous kit®(Ambion). The sample waspassed through the filter by centrifugation at ˜12,000 ×G for 1 min,then the filter was washed by the successive passage of three washsolutions through it. The collection tube was emptied between each wash,and each wash was passed completely through the filter at ˜12,000 ×G for1 min or longer, if required to pass all fluid. The first wash was with0.5 ml of 1.6 M guanidinium isocyanate (GuSCN)/70% ethanol, the last twowith 80% alcohol/0.1 M NaCl/4.5 mM EDTA/10 mM TrisHC1, pH 7.5. After thelast wash was passed through the filter, the filter was re-centrifugedover an empty collection tube to remove all traces of ethanol.

The sample was then eluted off the filter with 100 μl of 0.1 mM EDTA, pH8.0, which was applied directly to the filter at room temperature andcentrifuged through into a fresh collection tube. FIG. 1 and FIG. 2 showthe differences between preparations made from three different tissues,heart, brain, and liver, without and with the the pre-extraction step.It can be seen that, in either circumstance, a substantial portion ofthe let-7 miRNA is captured at 55% ethanol.

Example 2 Detection of miRNAs through Northern Blotting

For each RNA sample, 5 μl was combined with 5 μl of Gel Loading Dye II(Ambion). Prior to loading on a denaturing acrylamide gel, these sampleswere heated at 95° C. for 2-5 minutes. The standard gel was 15%acrylamide (monomer:bis ratio of 19:1), 7M urea, buffered with TBE(Tris-Borate-EDTA, Peacock and Dingman, 1967). The gel was routinelypre-run at 300-450 V for 30 minutes prior to loading the samples insample buffer, which also contained bromphenol blue and xylene cyanoltracking dyes. The electrophoresis was performed at 300-450 V for 45-60min, or until the bromphenol blue tracking dye was in the lower quadrantof the gel.

After electrophoresis, the gel apparatus was disassembled and the gelwas electroblotted to a BrightStar-Plus Nylon membrane (Ambion). Thisprocedure can be performed in a semi-dry apparatus using a stack ofthree sheets of Whatman filter paper (3 MM) soaked in 0.25×TBE above andbelow the gel sandwich at 200-400 mA for at least 0.2 A-hr. Extendingthis time does not lose sample. After blotting, the membrane was keptdamp and UV crosslinked using a commercial crosslinking device (theStratalinker™, Stratagene, Inc.)

The membrane was probed for the specific miRNA, let-7 (Pasquinelli etal., 2000) using an antisense probe that was 5′ end-labeled by T4Polynucleotide kinase.

In some cases other ubiquitous small RNAs were also probed for withantisense oligodeoxyribonucleotides at the same time. These included theU2 snRNA (Accession # X07913, complementary to the positions 28-42 ofthe 187 nt mouse U2 snRNA), U6 snRNA (Accession # V00853 or J00648,complementary to positions 83-103 of the 106 nt mouse RNA), and U43snoRNA (Accession # AJ238853, complementary to positions 20-37 of the 62nt human U43 RNA). All of these cross-hybridize readily between mouseand human. The procedure of Patterson and Guthrie (1987) was followedfor prehybridization, hybridization, and washing (Patterson and Guthrie,1987). The blots were prehybridized in {6×SSC, 10× Denhardt's solution,0.2% SDS} at 65° C. for at least one hour, then 10 ml of hybridizationsolution added {6×SSC, 5× Denhardt's, 0.2% SDS} which contained 5′end-labeled let-7, U43, U6, and/or U2 antisense oligodeoxynucleotideprobes (U43, U6, let-7 minimum=400,000 cpm; U2 minimum=200,000 cpm) andhad been filtered (0.45 μm pore) prior to use. Hybridization was for8-24 hr with agitation at room temperature. After hybridization,solutions were removed, and the blot washed 3 times for 5 minutes at RTwith wash solution: {6×SSC, 0.2% SDS}, then once with the same washsolution at 42° C. After the final wash, the blots were wrapped inplastic wrap and exposed to a phosphorimager screen (Molecular Dynamics)as per the manufacturer's instructions to quantify the amount of signalpresent in each band. The amount of let-7 in the fraction eluted wasoften compared to that in the flow-through, providing a “% bound”figure, as given in FIG. 1, FIG. 2, FIG. 3, FIG. 4, FIG. 5, and FIG. 6.For other figures, the amount of let-7 was compared to other samples orgiven as absolute. See specific examples.

For some of the examples, a second Northern blot was made from anagarose gel system to look for the presence of larger RNA species. Thesewere mRNAs for the ubiquitously-expressed genes cyclophilin(=Peptidylproline Isomerase or PPI), GAPDH and/or β-actin. The agarosegels were run and blotted using the NorthernMax Gly kit as described byAmbion. For probing the blot, antisense RNA probes were transcribed fromtemplates supplied by Ambion (cat#'s 7675, 7431, 7423) and hybridized inUltrahyb (Ambion), using all protocols as specified in Ambionliterature.

Example 3 Enrichment of Small RNA Molecules

Frozen mouse brain, heart, liver, and kidney were processed separatelyaccording to the following protocol for enrichment of small RNAs.

Approximately one-half gram of frozen mouse (strain Swiss-Webster, 6-12weeks old) tissue was crushed to fine powder under liquid nitrogen in amortar. This powder was further dispersed in standard lysis buffer (4 MGuSCN; 0.1 M beta-mercaptoethanol; 0.5% N-lauroyl sarcosine; 25 mMNa-citrate, pH 7.2) by the use of a rotor-stator homogenizer with a 7 mmgenerator at high speed for ˜30 sec.

After homogenization, 0.6 ml of the lysate was removed for this study.60 μl of 2M Na-acetate, pH 4.0, was added to the lysate, followedimmediately by 0.6 ml of acid phenol-chloroform. After 30 sec ofvigorous agitation, the aqueous phase was separated by centrifugation at16,000×G for 5 min. Four 100 μl aliquots of this aqueous phase were usedin four separate separations. The four aliquots had 100 μl of 40%, 50%,60%, and 70% ethanol added to each, then were passed through glass fiberfilters as in the RNAqueous procedure (Ambion). The 20%, 25%, 30%, and35% ethanol solutions that passed through these filters (theflow-through) was then adjusted to 55% ethanol final concentration bythe addition of 156, 133, 111, and 88.9 μl of ethanol, respectively. Allfour samples were passed over separate glass fiber filter columns. Thefilters were then washed with 0.7 ml of 4 M guanidinium isocyanate(GuSCN)/70% ethanol, followed by two washes with 0.5 ml 80% alcohol/0.1M NaCl/4.5 mM EDTA/10 mM TrisHCl, pH 7.5. After each wash was passedthrough the filter, the collection tube was emptied and replaced. Eachwash was passed through the filter by centrifugation as per theRNAqueous protocol (Ambion). filter re-centrifuged over an emptycollection tube to remove all traces of ethanol. The sample was theneluted off the filter with 100 μl of 0.1 mM EDTA, pH 8.0, which wasapplied directly to the filter at room temperature and centrifugedthrough into a fresh collection tube. The samples were examined byNorthern blot, as described, and compared on the same gel to anothersample that had been prepared from an equal volume of the same lysateusing the Totally RNA™ kit from Ambion.

Using both agarose and acrylamide Northern blots, the levels of theβ-actin, GAPDH, U2, and let-7 RNA species present in frozen mouse brain,heart, liver, and kidney were assayed in the material eluted from thefirst and second columns to determine the fraction recovered in thelatter. These are shown on FIG. 7. The larger mRNA is completely removedfrom the small-RNA enriched fraction.

FIG. 8 shows the relative enrichment of small RNAs using the methoddescribed in Example 3 as compared to the standard RNA isolation method.Here, samples of four common mouse tissues: brain, heart, kidney, andliver, were homogenized in standard lysis buffer as described inExample 1. After homogenization, two equal aliquots were taken of eachlysate. One was subjected to a standard RNA preparative procedure usingorganic extraction and ethanol precipitation, using 4 volumes of ethanolto precipitate to ensure full recovery of small RNA species. The otheraliquot was subjected to the enrichment procedure as described inExample 3. The concentration of RNA in each final sample was quantifiedusing absorbence at 260 nm. One microgram of each sample was separatedon a 15% denaturing polyacrylamide gel. This gel was electroblotted andthe resultant Northern blot probed for let-7 and U2 as described inExample 2. The amount of each probe hybridized to the appropriate areaof the blot was used to determine the relative amounts of each RNA inthe 1 μg samples. The signal for the enriched samples was divided by thesignal for the standard samples to provide the enrichment factors givenin FIG. 8. Enrichment in this case was from ˜3.5-8-fold by mass.

Example 4 Comparison to Standard Organic Extraction and EthanolPrecipitation

Samples from two mouse livers that had been stored frozen at ˜80 ° C.were ground to a fine powder under liquid nitrogen and homogenized in 10volumes (ml/g) the standard lysis buffer (4 M GuSCN; 0.1 Mβ-mercaptoethanol; 0.5% N-lauroyl sarcosine; 25 mM Na-citrate, pH 7.2)and then divided into four aliquots. One of the aliquots was extractedtwice with two different phenol-chloroform solutions as described in theTotally RNA™ protocol (Ambion), and the other three were subjectedindividually to the standard AMIK procedure. The RNA pelleted from theTotally RNA™ procedure was redissolved in 100 μl of 0.1 mM EDTA, pH 8.The final elution for the AMIK samples was in the same volume and samesolution. Samples were electrophoresed and blotted as described on both15% acrylamide and 1% agarose gels. The appropriate blots were probedfor β-actin, GAPDH, U2, U43, and let-7 as described. The recoveries ofeach RNA relative to the extraction procedure are summarized in thegraph in FIG. 9. The yield from the invention generated amounts of smallRNAs equal to or greater than the organic extraction procedure.

Example 5 Comparison to Standard Glass-Fiber Filter Purification

Frozen mouse liver and frozen mouse brain samples stored at ˜80° C. werehomogenized into standard lysis buffer at a ratio of 1 g tissue to 10 mlbuffer. After homogenization, all lysates were stored on ice until oneof two processing procedures was applied.

Starting with six aliquots of 100 μl from each parent lysate, 2 sampleswere processed by the RNeasy method from Qiagen, following their miniprocedure precisely after addition of 250 μl of the Tissue Lysis Buffer(TLB) supplied with the kit. The final four aliquots from each tissuewere prepared by the AMIK method previously described. The samples wereall eluted in 100 μl of water. For analysis, 5 μl of each of the sampleswere analyzed by electrophoresis on 15% acrylamide gels and blotting,and the blots were probed for let-7, U43, and U2. After usingphosphorimagery to quantify the bands, the signal levels were comparedbetween the methods for each small RNA. These results are shown in FIG.10. This invention was much more efficient at capturing all small RNAsthan the standard glass-fiber filter extraction procedure. Thisinability to capture small molecules with a standard procedure isaffected to some extent by the type of tissue as well, since the capturefrom liver lysate appears to be more efficient. This observation isconsistent with our observations using raw lysate (FIG. 1).

Example 6 Efficiency of Small RNA Recovery from Raw Lysate in ThreeDifferent Tissues

Samples of frozen heart, liver, and brain from mice (strainSwiss-Webster, 6-12 weeks old) were each pulverized under liquidnitrogen to a powder. This powder was weighed frozen and 10 ml of lysisbuffer per gram of tissue was added (weights ranged from 200 to 500 mg).Samples were homogenized with a rotor-stator homogenizer immediatelyafter addition, then divided into 8×100 pi aliquots on ice. To these,53.9, 66.7, 81.8, 100, 122.2, 150, 185.7, and 233.3 pi of absoluteethanol were added to make final concentrations of 35, 40, 45, 50, 55,60, 65, and 70% ethanol. Each of these was passed over a glass fiberfilter column as found in the RNAqueous® kit (Ambion), and theflow-through from this collected. The RNA in the flow-through wasphenol-chloroform extracted and ethanol precipitated with four volumesof ethanol to ensure precipitation of small RNAs. After pelleting theRNA by 30 min of centrifugation at 16,000×G, the pellet was washed oncewith 80% ethanol and then redissolved in 60 μl of 0.5 mM EDTA, pH 8.0.The filters were washed three times, once with 0.7 ml of 4 M guanidiniumisocyanate (GuSCN)/70% ethanol, followed by two washes with 0.5 ml 80%alcohol/0.1 M NaCl/4.5 mM EDTA/10 mM TrisHCl, pH 7.5. Each wash wasperformed as above, by centrifugation at 12,000×G for 1 min orsufficient time to clear all liquid through the filter, with collectiontubes emptied after each. Samples were eluted using 2 separate additionsof 30 μl of 0.1 mM EDTA, pH 8.0 which was applied directly to the filterafter pre-warming to 95° C., each centrifuged through into the samefresh collection tube. Equal amounts (5 μl) of both thefilter-bound-and-eluted and the flow-through were analyzed by Northernblot as described above. Since bound and flow-through were on the sameblot, the amount of let-7 RNA bound could be calculated for each ethanolconcentration with each tissue. This data is plotted in FIG. 1 and FIG.2. It is apparent that the binding behavior for each tissue wasdifferent, in terms of the concentration of ethanol required toimmobilize all let-7 RNA on the glass fiber filter. However, the maximumappears to be achieved for all tissues by 55% ethanol.

Example 7 Purification from Cultured Cells

Cells were collected from two lines, HEK-293 (derived from humanembryonic kidney) or HeLa (human cervix) cells, from culture flasks bytrypsinization. After counting, these cells were added at a level ofabout one million each to two 2 ml microcentrifuge tubes and pelleted bycentrifugation. Supernatant was removed and the pelleted contents ofeach tube was resuspended in 700 μl of lysis buffer as described in thestandard procedure (Example 1). The cells were lysed by agitating thetube vigorously for 30 sec rather than use of a homogenizationapparatus. For each set, one set was immediately made about 55% ethanolby addition of 860 μl absolute ethanol. The other aliquot was processedas stated in the standard procedure: acidified by the addition of 70 μl2 M Na-Acetate buffered to pH 4, followed by extraction with 700 μl acidphenol-chloroform, then addition of 860 μl ethanol to the recoveredupper phase. Both samples were passed through glass-fiber filters,washed three times, and eluted with 100 μl 0.1 mM EDTA, pH 8 asdescribed above. Five μl of each eluate was electrophoresed on a 15%acrylamide gel and Northern blotted for U2 and let-7. The levels ofeach, as determined from phosphorimagery of the blot, are shown in FIG.11. The recovery of small RNAs from all the methods appears good, butrecovery from HeLa cells was enhanced by the pre-extraction procedure.

Example 8 Pre-extraction using Different Salt Conditions

Frozen mouse liver was homogenized into Lysis Buffer at 1.1× normalconcentration minus Na-citrate (4.4 M GuSCN; 0.11 M β-mercaptoethanol;0.55% N-lauroyl sarcosine). Immediately after homogenization, two 1.8 mlaliquots were removed from this lysate and 200 μl of 0.25M Na-citrate ateither pH 7.2 or 4.5 was added to each. Four 400 μl aliquots wereremoved from these 2 ml portions, and 40 μl of either water, 1M, 2M, or3M NaOOCCH₃ (sodium acetate, pH 4.5) was added to each of these, to givea final [NaOOCCH₃] of zero and about 0.1, 0.2, and 0.3 M. The sampleswere each extracted with 440 μl of acid phenol-chloroform and 300 μl ofthe upper phase recovered. This was made 55% in ethanol by the additionof absolute ethanol and purified over a glass-fiber filter column asdescribed in the standard procedure. Each sample was applied to a 15%acrylamide gel, blotted and probed as described above. The levels of U2,U43, and let-7 determined for each are shown in the FIG. 12 graph. Yieldappears to be roughly equivalent at both pH's (although U43 wasvariable), but the best yield appears in the presence of 0.2 M NaOOCCH₃at both pH's.

Example 9 Binding of Small RNA Molecules at Different Guanidinium andEthanol Concentrations

Mouse liver was homogenized in standard lysis buffer and extracted withacid phenol-chloroform. The extracted lysate was divided in twoportions. An equal volume was added to each consisting of either LysisBuffer with no guanidinium (0.1 M β-mercaptoethanol; 0.5% N-lauroylsarcosine; 25 mM Na-citrate, pH 7.2) or Lysis Buffer with 2 M GuSCN (2 MGuSCN; 0.1 M beta-mercaptoethanol; 0.5% N-lauroyl sarcosine; 25 mMNa-citrate, pH 7.2), creating solutions with a final [GuSCN] of 2 M and3 M, respectively. These were then further divided into 18 aliquots of200 pi each, and ethanol additions made in one of two manners. The firstmethod was the addition of 22.2, 35.3, 50, 66.7, 85.7, 107.7, 133.3, and200 pi of absolute ethanol. This gave final ethanol concentration of 10,15, 20, 25, 30, 35, 40, 45, and 50%, with corresponding finalguanidinium concentrations that decreased with increasing ethanol. (2.7,2.55, 2.4, 2.25, 2.1, 1.95, 1.8, 1.65, and 1.5 M for 3 M initialconcentration; 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, and 1.0 M for 2 Minitial concentration). The second method added equal volumes of ethanolsolutions in water from 20-100% to give the same final ethanolconcentrations, but with consistent guanidinium concentrations of 1.5 or1 M within each series. After ethanol addition, each of these sampleswas bound to the glass-fiber filter and the standard procedure wasfollowed. Samples were run on both acrylamide and agarose gels to assayfor the presence of β-actin, GAPDH, PPI (cyclophilin), U2, and let-7.The binding behavior of each species as ethanol concentration increasedwas plotted for the four series in FIG. 3, FIG. 4, FIG. 5, and FIG. 6.From these series, it is demonstrated that differences exist in thebehavior of the differently-sized RNA species, such that by manipulatingboth salt and ethanol concentration the binding of quite restricted sizeranges of RNA molecules can be achieved, indicating more refinedsize-fractionation procedures can be performed.

Example 10 Use of Small RNAs to Probe Microarrays

Small RNAs enriched using procedures described in Examples 3 or 9 may beused in the microarray technologies described in the specification. Inone example, the probes affixed to the microarray may contain sequencesspecifically designed to capture known miRNAs or siRNAs. Alternatively,the probes affixed to the microarray could be mRNA sequences to look forpotential in vivo biological targets for miRNAs or siRNAs. The small RNAmolecule population could be labeled radioactively or with tags that arereactive to light or able to bind secondary molecules capable ofreacting with light. These direct or indirect labels could be attachedthrough enzymatic means well-known to those of skill in the art such as:removal of the 5′ phosphate with phospahtase followed by addition ofmodified phosphate with polynucleotide kinase; or addition to the 3′ endof one or several tagged nucleotides with RNA ligase or polymerases suchas poly-A polymerase.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references are specifically incorporated herein byreference.

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1.-53 (canceled)
 54. A method for isolating small RNA molecules havingat most 100 nucleotides or fewer from a sample comprising: a) lysingcells in a lysing solution to produce a lysate; wherein the lysingsolution comprises a chaotropic agent; b) adding to the lysate anethanol solution to form a lysate/ethanol mixture; c) applying thelysate/ethanol mixture to a first solid support and collectingflow-through lysate/ethanol mixture; d) adding to the flow-throughlysate/ethanol mixture of step c) an ethanol solution; e) applying thelysate/ethanol mixture of step d) to a second solid support; and f)eluting small RNA molecules from the solid support, wherein thelysate/ethanol mixture applied to the first solid support is betweenabout 20% to about 35% ethanol and wherein the lysate/ethanol mixtureapplied to the second solid support is between about 35% to about 70%ethanol.
 55. The method of claim 54, wherein the lysing solutioncomprises a detergent.
 56. The method of claim 55, wherein theconcentration of the detergent in the lysing solution is about 0.1% toabout 2%.
 57. The method of claim 54 wherein the chaotropic agent is aguanidinium salt and/or wherein the detergent is N-lauroyl sarcosine.58. The method of claim 55 wherein the chaotropic agent is a guanidiniumsalt and/or wherein the detergent is N-lauroyl sarcosine.
 59. The methodof claim 56 wherein the chaotropic agent is a guanidinium salt and/orwherein the detergent is N-lauroyl sarcosine.
 60. The method of claim54, wherein the lysate is extracted with phenol and/or chloroform. 61.The method of claim 55, wherein the lysate is extracted with phenoland/or chloroform.
 62. The method of claim 57, wherein the lysate isextracted with phenol and/or chloroform.
 63. The method of claim 54,wherein the lysing solution comprises a buffer, preferably in aconcentration of about 10 mM to about 300 mM.
 64. The method of claim60, wherein the lysing solution comprises a buffer, preferably in aconcentration of about 10 mM to about 300 mM.
 65. The method of claim54, further comprising washing the solid support with a first washsolution after applying the lysate to the solid support and optionallywashing the solid support with a second wash solution after washing withthe first wash solution.
 66. The method of claim 65, wherein the firstwash solution comprises a chaotropic agent, preferably wherein thechaotropic agent is a guanidinium salt and the first wash solutionfurther comprises alcohol.
 67. The method of claim 66, wherein thesecond wash solution comprises alcohol.
 68. The method of claim 54,wherein the small RNA molecules are eluted from the solid support at atemperature of about 60° C. to about 100° C.
 69. The method of claim 57,wherein the small RNA molecules are eluted from the solid support with alow-ionic-strength solution.
 70. The method of claim 54, wherein thesolid support comprises silica, in particular glass fiber.
 71. Themethod of claim 54, wherein the first and the second solid support aremade of the same material.